Suppressing Pyroptosis Augments Post-Transplant Survival of Stem Cells and Cardiac Function Following Ischemic Injury

Figures

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  Figure 1. Macrophages are recruited in rat hearts with ischemia/reperfusion (I/R) injury. (A) Cardiac fibrosis occurred in the hearts with I/R injury after a week. Cardiac fibrosis was measured by Masson’s trichrome staining using paraffin section. Blue indicates that collagen expression is increased in the infarct zones of the hearts with I/R injury (left). Recruited macrophages are revealed by immunofluorescence staining in the hearts with I/R injury (right). Antibodies indicate the following: red: cTnT, a specific cardiomyocyte marker; green: F4/80, a specific macrophage marker; and blue: DAPI, a nuclear marker. Scale bar = 50 µm, n = 5 per group. (B) F4/80 expression was detected in the hearts one day after I/R injury. The heart sections from the acute I/R injury areas were analyzed by immunofluorescence staining with F4/80 (green: macrophages) using a frozen section. DAPI indicates nuclear staining. Scale bar = 100 μm. n = 3 per group (C) Quantitative analysis was performed for F4/80-positive cells in the heart sections. F4/80-positive cells in the border zone and infarct zone were significantly increased. * p < 0.05 vs remote zone (D) IL-1β expression levels were further analyzed by…

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  Figure 2. Expression of pyroptosis-related genes and proteins is increased in the heart with ischemia/reperfusion injury. (A) Pyroptosis-related gene expression was measured by qRT-PCR. mRNA was extracted from the infarct zones of the hearts. The expression of pyroptosis-related genes, including caspase-1, caspase-11, GSDMD, IL-1β, and IL-18, was significantly increased in the early I/R-injured hearts. CASP1 indicates caspase-1. CASP11 indicates caspase-11. GSDMD indicates gasdemin D. # p < 0.05 vs. normal, ** p < 0.01 vs. normal, * p < 0.001 vs. normal, (B) Caspase-1, caspase-11, GSDMD, and IL-1β expression levels in normal and I/R-injured hearts were detected by western blotting. (C) Pyroptosis-related proteins expression was normalized to β-actin. ** p < 0.01 vs. normal, * p < 0.001 vs. normal, (D) Caspase-1, caspase-11, GSDMD, and IL-1β mRNA expression levels by qRT-PCR were higher in the border zone and infarct zone compared than in the remote zone a day after I/R injury in the hearts. Pyroptosis-related gene expression was normalized to GAPDH. # p < 0.05 vs. normal, ** p < 0.01 vs. normal, * p < 0.001 vs. normal. (E) Caspase-1, caspase-11, GSDMD, pro-IL-1β, and cleaved IL-1β protein…

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  Figure 3. Expression of pyroptosis-related genes is increased by M1 macrophage conditioned media in ASCs. hASCs were incubated with M1-like macrophage-CM (MØCM) for 24 h. (A) mRNA and (B,C) protein expression levels were measured by qRT-PCR and western blotting, respectively. # p < 0.05 vs. Control, ** p < 0.01 vs. Control, * p < 0.001 vs. Control. (D) Caspase-1 and GSDMD and (E) Caspase-4 and IL-1β expression levels were detected by immunofluorescence. Blue indicates the nuclei. Scale bar = 50 µm.

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  Figure 4. IL-1β plays an important role for pyroptosis induction in hASC. IL-1β recombinant protein at concentrations of 1 ng/mL and 10 ng/mL was used to treat hASCs for 24 h. Then, mRNA and protein levels were detected by (A) qRT-PCR and (B) western blotting. # p < 0.05 vs. Control, ** p < 0.01 vs. Control, * p < 0.001 vs. Control. hASCs were cultured for 24 h with IL-1β siRNA-transfected macrophage-CM (Si-IL1β_MØCM). Then, pyroptosis-related mRNA and protein levels were detected by (C) qRT-PCR and (D) western blotting, respectively. # p < 0.05 vs. MØCM, * p < 0.001 vs. MØCM.

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  Figure 5. Screening miRNAs predicted to target IL-1β. miRNAs (13 miRNAs) were selected based on (A) 4 databases, miRanda, RNA22, miRwalk, and TargetScan (#1), and DEG analysis with criteria of p < 0.05 and a fold change > 2 resulted in the final selection of miRNA-762 (#2). (B) IL-1β protein levels in hASCs treated with miRNA-762 mimic (miRNA-762), negative control (N.C.) or anti-miRNA-762 (Anti-762) (# p < 0.05, ** p < 0.01 vs. Normal). (C) miRNA-762 levels were quantified by DEG analysis for the normal group and the ischemic heart group. (D) Schematic representation of the matched miRNA-762 and 3′ UTR of IL-1β mRNA. (E) Luciferase activity of miRNA-762-transfected cells was measured in response to the 3′UTR of IL-1β. miRNA-762 or negative control (N.C.) was co-transfected into HeLa cells with the pmir-GLO vector or pmir-GLO containing the 3′ UTR of IL-1β as a miRNA-762 binding site. Luciferase activity was normalized to the renilla activity. (** p < 0.01 vs. Control).

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  Figure 6. hASCmiR−762 affects the reduction of fibrosis at ischemia/reperfusion injury heart. (A) Seven days after hASC or hASCmiR−762 transplantation, PKH26-stained ASCs were detected in hearts one week after I/R-injury. Scale bar = 100 μm. (B) PKH26-positive cells were quantified by counting stained cells and averaged per section. # p < 0.05 (C,D) Fibrosis was detected by Masson’s trichrome staining in three hearts of rats per group at one week after I/R injury. # p < 0.05 vs. I/R group, ** p < 0.01 vs. I/R group. n = 3 per group. (E–H) Left ventricular function was measured at 3 weeks after I/R injury and hASCmiR−762 transplantation. Each component of cardiac function was quantified as follows. n = 3 per group. (E) heart rate, (F) stroke volume, (G) ejection fraction, (H) stroke work. Each experimental group was measured five times. # p < 0.05, ** p < 0.01, * p < 0.001.